The long-range goal of this project is to understand the upstream elements coordinating signaling through Ras and c-Raf-1 at the plasma membrane. Our and other recent investigations have identified Kinase Suppressor of Ras (KSR) as a component of this cascade, although its exact function remains controversial. Our hypothesis is that KSR phosphorylates and activates c-Raf-1 and that this event is crucial for Ras-mediated oncogenesis. During the last funding period, we generated new tools to address these issues. We developed a two-stage in vitro KSR activity assay and a KSR knockout (KO) mouse that are essential for the studies to be implemented in this proposal. In Specific Aim I, we will examine mechanisms by which upstream effector kinases link KSR to Tyr kinase pathways signaling through Ras. In particular, we will evaluate Akt as a transmodulator of epidermal growth factor receptor (EGFR) function as preliminary data suggest Akt directly phosphorylates and activates KSR. In Specific Aim II, we will investigate the relevance of KSR to oncogenesis through the EGFRIRas connection using transplantable tumor models and cell lines from the KSR KO mouse. Preliminary data suggest that dominant negative KSR reverts the transformed phenotype of A431 cells which are driven through the EGFR. Specific Aim Ill will extend studies to transgenic models of Ras-/Akt-mediated tumorigenesis. For these studies, KSR+/+ and KSR-/- animals will be crossed with transgenic Ha-Ras models and with PTEN KO mice. These models manifest spontaneous tumor formation. Preliminary studies with TC.AC mice suggest KSR may be necessary for optimal Ras-mediated skin tumorigenesis. The studies proposed in this application have the potential to resolve an enigma in the field of Ras/c-Raf-1 signaling, It is generally accepted that binding of Ras to c-Raf-1 is insufficient for c-Raf-1 activation, and that other membrane-associated events are necessary. Our studies may offer new insight into this issue. Concomitantly, these investigations may identify new elements of Ras signaling of oncogenesis. The use of our genetic tools is likely to lead to definitive information on this signaling. A more precise ordering of these events might provide future opportunities for pharmacological modulation of these pathways.